Methods of treating prader-willi syndrome

ABSTRACT

The present invention relates to methods for regulating prohormone convertase (PC1) and compounds and treatments which increase PC1 levels, for treating Prader-Willi Syndrome (PWS).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 16/306,233, filed Nov. 30, 2018, which is a 371U.S. National Stage application of International Patent Application No.PCT/US2017/035655, filed Jun. 2, 2017, which claims priority to U.S.Provisional Patent Application No. 62/345,133 filed Jun. 3, 2016; U.S.Provisional Application No. 62/375,662 filed Aug. 16, 2016, each ofwhich is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant DK052431awarded by the National Institutes of Health (NIH). The government hascertain rights in the present invention.

FIELD OF THE INVENTION

The present invention relates to methods for regulating prohormoneconvertase (PC1) and compounds and treatments which increase PCI levels.

BACKGROUND OF THE INVENTION

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressedgenes in an imprinted region of chromosome 15q. Among the canonicalphenotypes are hyperphagic obesity, central hypogonadism and low growthhormone. Rare microdeletion PWS patients define a 91 kb minimum criticaldeletion region encompassing 3 genes, including the non-coding SNORD116.We have found that NHLH2 and PC1 are downregulated in PWS iPSC-derivedneurons as compared to unaffected controls. Nhlh2 and Pcsk1 transcriptlevels are reduced in hypothalami of fasted Snord116^(p−/m+) mice.

Deficiency of Nhlh2 in mice causes obesity, hypogonadism, and growthfailure. Nhlh2 promotes expression of the prohormone convertase, (PC1).Humans and mice deficient in PC1 display hyperphagic obesity,hypogonadism, decreased growth hormone, and diabetes due to impairedprohormone processing. For example, Snord116^(p−/m+) mice display invivo functional defects in prohormone processing of proinsulin, proGHRH,and proghrelin associated with reductions in PC1.

Currently there are no treatments for PWS patients and effectivetreatments and model systems are urgently needed.

SUMMARY OF THE INVENTION

The methods of the present invention provide for regulating prohormoneconvertase by administering an effective amount of a phosphodiesterase 4inhibitor (PDE4 inhibitor or PDE4i). Expression of the prohormoneconvertase may be upregulated by administration of a therapeuticallyeffective amount of a PDE4 inhibitor. The PDE4 inhibitor may beadministered to a cell or to a patient with Prader-Willi syndrome. ThePDE4 inhibitor may be administered to an obese subject. It is alsoexpected that these methods will be useful for treating patients withSchaaf-Yang Syndrome and Autism Spectrum Disorder.

The PDE4 inhibitor may be administered orally, intravenously,subcutaneously, intrathecally, topically, intranasally, or to the lungs.

The PDE4 inhibitor can include, theophylline, roflumilast, apremilast,ibdulast, GSK356278, MK0952, IBMX as well as combinations of thesedrugs.

In certain embodiments, the PDE4 inhibitor can include any of theinhibitors from Tables 1A or Table 1B.

In additional embodiments, combinations of PDE4 inhibitors may be usedin the present methods.

The methods of the present invention also include administering atherapeutically effective amount of an adenylate cyclase activator. Theadenylate cyclase activator can be administered to a cell or to apatient with Prader-Willi syndrome. The adenylate cyclase activator maybe administered to an obese subject.

The adenylate cyclase activator can be administered orally,intravenously, intrathecally, intranasally, topically, or to the lungs.The adenylate cyclase activator can include, Forskolin, FD1, FD2, FD3,FD4, FD5 (NKH477), FD6 as well as combinations of these drugs.

The PDE4 inhibitor may also be administered together with the adenylatecyclase activator.

The methods of the present invention also include administering atherapeutically effective amount of an MC4R agonist. The MC4R agonistcan be administered to a cell or to a patient with Prader-Willisyndrome. The MC4R agonist may be administered to an obese subject.

The MC4R agonist can be administered orally, intravenously,intrathecally, intranasally, topically, or to the lungs. The MC4Ragonist can include RM-493 (Setmelanotide), TTP2515, 2-aminothiazolederivatives, MK-0493, and combinations thereof. The MC4R agonist can beadministered in combination with the PDE4 inhibitors and/or adenylatecyclase activators described herein.

The methods of the present invention also include methods wherein theadministration results in one or more of the following improvements inthe patient: decreases or ameliorates hyperphagia; increases PCSK1levels; increases PC1 level and/or activity; decreases circulatingproinsulin to insulin ratio, thus increasing insulin secretion;decreases circulating proghrelin to ghrelin ratio; decreases circulatingPOMC to ACTH ratio; amelioration of hypothyroidism, decreasescirculating ratio of pro-oxytocin to oxytocin, thus increasing oxytocinproduction in the brain and increases alpha-MSH production in the brain;decreases circulating ratio of pro-BDNF to BDNF (increase brain levelsof BDNF); and increases the ratio of prohormone: hormone (decreasespro-mature hormone); wherein the symptom, levels, or ratios are inreference to the patient's disease symptom, levels, or ratios.

In certain embodiments, the methods provide for treating Prader-WilliSyndrome (PWS) comprising administering a phosphodiesterase 4 inhibitor(PDE4i) to a subject in need thereof, thereby alleviating, eliminatingor preventing one or more symptoms of PWS.

In certain embodiments, administering the PDE4i upregulates cyclicadenosine monophosphate (cAMP) concentrations or activity in thesubject.

In certain embodiments, PWS is characterized by decreased expression ofNHLH2.

In additional embodiments, decreased expression of NHLH2 results indecreased expression of PCSK1.

In certain embodiments, increasing concentrations or activity of cAMPupregulates expression of Pcsk1.

In additional embodiments, the PDE4i is a selective PDE4i. In additionalembodiments, the PDE4i is a non-selective PDE4i.

In certain embodiments, the selective PDE4i is selected from AN2728,apremilast, cilomilast, diazepam, ibudilast, luteolin, mesembrenone,piclamilast, roflumilast, rolipram, E6005, GSK356278 and MK0952.

In certain embodiments, the non-selective PDE4i selected from methylatedxanthines and derivatives thereof, caffeine, aminophylline,3-isobutyl-1-methylxanthine, paraxanthine, pentoxifylline, theobromine,and theophylline.

In yet additional embodiments, the one or more symptoms includehyperphagia, reduced metabolic rate, obesity, hypogonadism,hypoadrenalism, decreased growth hormone production, poor muscle tone,sleep disorders, gastrointestinal disorders, reduced stamina, reducedability to focus, impaired cognition, behavioral disorders, anxiety,growth failure, reduced conversion of immature hormones to mature andactive forms, and diabetes mellitus and diabetes insipidus.

In certain embodiments, the method further comprises administering oneor more additional therapeutic agents effective for treating oralleviating one or more symptoms of PWS.

In certain embodiments, the immature hormones comprise one or more ofinsulin, ghrelin, GHRH, alpha-MSH, oxytocin, orexin, BDNF, vasopressin,NPY, AGRP, and gonadotropins, ACTH.

In certain embodiments, the one or more additional therapeutic agentseffective at treating or alleviating PWS include insulin, an insulinreceptor agonist, ghrelin, a ghrelin receptor agonist, GHRH, a GHRHreceptor agonist, alpha-MSH, an alpha-MSH receptor agonist, oxytocin, anoxytocin receptor agonist, orexin, an orexin receptor agonist, BDNF, aBDNF receptor agonist, vasopres sin, a vasopres sin receptor agonist,NPY, an NPY receptor agonist, AGRP, an AGRP receptor agonist,gonadotropin, a gonadotropin receptor against, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a model showing how deficiencies in Nhlh2 and PC1 may drivethe major neuroendocrine phenotypes of PWS. A deficiency in prohormoneprocessing owing to deficits in PC1 and Nhlh2 production may explainmany of the major neuroendocrine phenotypes of PWS. It is hypothesizedthat paternal loss of SNORD116 may be sufficient to cause deficienciesin Nhlh2 and PC1, in turn causing functional defects in prohormoneprocessing. Arrows/lines that are dashed indicate theoreticalconnections. Arrows/lines that are solid indicate pathways that havebeen investigated.

FIG. 2 is a schematic showing the rationale for treatment of PWSutilizing agents that increase cellular cAMP levels in order to increaselevel and/or activity of cellular PC1 and increase prohormoneprocessing.

FIGS. 3A-P are graphs showing downregulation of PC1 in PWS models isassociated with impaired prohormone processing; PCSK1 transcript levelscan be increased in unaffected control by treatment with Forskolin.

FIG. 4 is a schematic showing the therapeutic rationale forco-administration of MC4R agonists and/or AgRP inhibitors in combinationwith Forskolin and/or Theophylline in individuals with PWS and possiblyother types of obesity, including common obesity.

FIGS. 5A-H are graphs showing that the application of Forskolin, an ACagonist, elevates PCSK1/Pcsk1 transcript levels in primary mouseneurons, iPSC-derived neurons, and primary mouse pancreatic islets.

FIGS. 6A-F are graphs showing that application of phosphodiesteraseinhibitors to iPSC-derived neurons increases PCSK1 transcript levels andprohormone processing. FIG. 6A: Theophylline increases PCSK1 transcriptlevels in D34 iPSC-derived hypothalamic ARC neurons (1023A line) at 10mM concentration. FIGS. 6B-C: Roflumilast increases PCSK1 transcriptlevels in iPSC-derived neurons at Day 40 of differentiation (1043D3line) at 1 mM concentration. FIGS. 6D-E: Combination treatment withRoflumilast (100 nM) and Forskolin (1 μM) increase PCSK1 transcriptlevels and increase POMC processing to ACTH at lower concentrations thaneither agent alone, suggesting an additive or possibly synergisticeffect. FIG. 6F: MK0952 applied at 10 μM in combination with 1 μMForskolin increases PCSK1 transcript levels ˜2-fold in iPSC-derivedneurons (1043D3 line).

FIGS. 7A-B are graphs showing that a single treatment with MK0952increases hypothalamic Pcsk1 in wild type mice. FIG. 7A is a graphshowing that body weights of all mice used were comparable. FIG. 7B is agraph showing that MK0952 administered by oral gavage in 10% methylcellulose at a dose of 10 mg/kg body weight increased hypothalamic Pcsk1levels by about 25%. Treatment with Forskolin at 25 mg/kg did not resultin increased hypothalamic Pcsk1 levels. Combination treatment withMK0952 (10 mg/kg) and Forskolin (25 mg/kg) also resulted in a 25%increase in hypothalamic Pcsk1 transcript levels, likely due largely tothe effects of MK0952.

FIGS. 8A-C are schematics, tables and graphs showing aspects of theclinical trial design.

DETAILED DESCRIPTION

The present disclosure provides for methods to regulate PC1 (prohormoneconvertase 1) levels in vitro or in vivo. The methods can be used toupregulate (increase expression) or increase PCI levels and/or activity.Also encompassed by the present disclosure are methods to treatPrader-Willi syndrome (PWS) and other forms of obesity. The methods maycomprise the step of administering a therapeutically effective amount ofa PDE4 inhibitor and/or an adenylate cyclase activator. It is alsoexpected that these methods will be useful for treating patients withSchaaf-Yang Syndrome and Autism Spectrum Disorder (Fabienne SchallerFrançoise Watrin Rachel Sturny Annick Massacrier Pierre SzepetowskiFrançoise Muscatelli; Hum Mol Genet (2010) 19 (24): 4895-4905. Green L,Fein D, Modahl C, Feinstein C, Waterhouse L, Morris M. Oxytocin andautistic disorder: alterations in peptide forms. Biol Psychiatry. 2001Oct. 15; 50(8):609-13.

Theoretical mechanisms to increase cellular prohormone convertase 1levels/activity include but are not limited to: (1) upregulation at thetranscript level by engaging endogenous promoters, (2) directlyincreasing enzymatic activity of PC1, (3) increasing rates oftranslation of PCSK1 to PC1, (4) decreasing degradation of PC1enzyme/protein, one possible approach is by decreasing levels (viaantisense oligo “genetic knockdown,” traditional small moleculeinhibition, or other) of the endogenous inhibitor of PC1, ProSAAS, (5)decreasing degradation (miRNA targeted, non-sense mediated decay,putative mRNA methylation levels) of PCSK1 transcript, therebyincreasing translation, (6) PC1 itself is processed from a 92 kDazymogen to a 66 kDa mature enzyme, thus increasing levels of preproPC1processing could also have therapeutic utility, and (7) delivery ofadditional PCSK1 cDNA to the cell by gene therapy methods, (8) deliveryof SNORD116 RNAs by gene therapy methods, and (9) direct delivery of thePC1 enzyme into the circulation and/or tissues with enzyme replacementtherapies.

The present findings suggest that the major neuroendocrine features ofPWS are likely due to functional PCI deficiency. See FIG. 1. As the geneencoding PC1, PCSK1, is intact in PWS, increasing the levels of PC1expression and/or activity in PWS patients will correct this functionalPC1 deficiency. Pharmacologically, this increase in PC1 levels can beachieved by administration of agents that increase cyclic adenosinemonophosphate (cAMP) levels or block cAMP degradation.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis ofcyclic AMP and cyclic GMP, thereby regulating the intracellularconcentrations of these cyclic nucleotides, their signaling pathwaysand, consequently, a myriad of biological responses in health anddisease. Maurice et al. Advances in targeting cyclic nucleotidephosphodiesterases. Nat. Rev. Drug. Discov. 13(4):290-314 (2014). PDE4isoforms are highly expressed in cells that regulate immunoinflammatoryresponses and tissue remodeling. Id. Inhibition of PDE4 results in anincrease in cAMP levels in the cell. A large number of PDE4 inhibitorsare available. Non-limiting examples of PDE4 Inhibitors include:Theophylline, Roflumilast, Apremilast, Ibudilast, GS K356278, MK0952,IBMX (3-isobutyl-1-methylxanthine), Mesembrenone, Rolipram, Piclamilast,Luteolin, Drotaverine, AN2728, Cilomilast, Diazepam, Luteolin, andE6005. Other phosphodiesterase inhibitors include, methylated xanthinesand derivatives (such as caffeine, aminophylline, paraxanthine,pentoxifylline, theobromine, and theophylline).

The levels of cAMP may also be increased using agents which activateadenylate cyclase. Non-limiting examples of adenylate cyclase activatorsinclude: Forskolin, FD1, FD2, FD3, FD4, FD5 (NKH477), and FD6.

PDE4 inhibitors and adenylate cyclase activators can be referred toalone or in combination as therapeutic agents.

TABLE 1A Selected PDE4 Inhibitors PDE4 Brain Drug selectivitypenetration Status Theophylline nonselective Good Generic RoflumilastSelective limited Approved COPD: Takeda/AstraZeneca (LOE: 2020/2031)Apremilast Selective None approved psoriasis: Celgene (LOE: 2028)Ibudilast nonselective Yes approved in Japan Phase 2 2015 ALS byMedicinova in US GSK356278 Selective Good suspended: GSK Phase 1 2012Huntington's Disease MK0952 selective yes suspended: Merck Phase 2 2007Alzheimer's Disease; See: Heckman P R, Wouters C, Prickaerts J.Phosphodiesterase inhibitors as a target for cognition enhancement inaging and Alzheimer's disease: a translational overview. Curr Pharm Des.2015; 21(3): 317-31. Review. PubMed PMID: 25159073. Gallant M, et al.Discovery of MK-0952, a selective PDE4 inhibitor for the treatment oflong- term memory loss and mild cognitive impairment. Bioorg Med ChemLett. 2010 Nov. 15; 20(22): 6387-93. doi: 10.1016/j.bmcl.2010.09.087.Epub 2010 Sep. 21. PubMed PMID: 20933411. IBMX nonselective n/alaboratory use only

TABLE 1B Compounds useful as PDE Inhibitors (PDEi) GEBR-32a AVE 8112ABAY 60-7550 Rolipram GRC-4039 Anagrelide GEBR-7b Revamilast CilostazolSelaginpulvilins K DG 071 Milrinone Selaginpulvilins L MEM 1414Olprinone GSK256066 Mesopram Parogrelil Chlorbipram SH 636 PimobendanFFPM ZK 117137 Ibudilastroflumilast Cilomilast MEM-1018 Ro 20-1724Piclamilast MEM-1091 CDP840 BC8-15 MEM-1917 Tofimilast ZL-n-91 R 1627Oglemilast NIS-62949 AV-11 Tetomilast CHF6001 AV-411 Lirimifast4-(8-(3-Fluorophenyl)-1,7- Eyevinal Sildenafil naphthyridin-6-yl)transcyclohexanecarboxylic Acid 4,5,6,7-tetrahydro-1H-1,2- IbinalTadalafil diazepin-7-one derivatives PDE-310 KC-404 Vardenafil RPL554Ketas Udenafil L-454,560 MN-166 Avanafil GS-5759 Pinatos DipyridamoleBPN14770 D159687 E-4010 TAK-648 D159797 E-4021 PF-02545920 BPN14770E-8010 RO5545965 PF-00489791 Zaprinast AMG 579 PF-04447943 Iodenafil TAK063 ABI-4 Mirodenafil PF-05180999 Crisaborole DA-8159 BCA909 FCPR03 BAY73-6691 HT-0712 A-33 PF-2545920 Ro-20-1724 T-094 SCH-1518291 ME 3167Resveratrol 4-amino substituted condensed pyrimidine compounds as PDE4inhibitors (See: U.S. Pat. No. 9,505,724 B2) ZK 62711 ITI-214Substituted pyridine and pyrazine compounds as PDE4 inhibitors (See U.S.Pat. No. 9,573,937 B2) APTA-2217 Cilostazol Azabenzimidazole compounds(See U.S. Pat. No. 9,120,788 B2) B 9302-107 Milrinone phosphodiesterasetype 4 inhibitors, containing 2-phenyl-4- oxazole moiety, exemplified by“compound 4C” from Li Y S et al Design, synthesis and biologicalevaluation of 2,4-disubstituted oxazole derivatives as potential PDE4inhibitors. Bioorg Med Chem. 2017 Mar. 15; 25(6): 1852- 1859. doi:10.1016/j.bmc.2017.01.047. Epub 2017 Feb. 3. BY 217 Enoximone Heteroarylinhibitors of PDE4 - as described in: WO2015048407A1. BYK 20869 ASP9831Daliresp Tadalafil Daxas Sildenafil IN-ALR 01 AN2898 Libertek AN2728 DNS001 vinpocetine AVE 8112 erythro-9-(2-hydroxy- 3-nonyl)adenine (EHNA)

TABLE 2 Selected Adenylate Cyclase Activators Brain Drug AC isoformsactivated penetration Status Forskolin All except AC9 Yes OTC FD1 AC2(AC2 expressed in brain, Not tested Synthesized by Nippon Kayakuskeletal muscle, lung, heart ⁷) Co. only tested in cell-based models,not FDA-approved FD2 AC2 Not tested Synthesized by Nippon Kayaku Co.only tested in cell-based models, not FDA-approved FD3 AC3 (AC3expressed in brain, Not tested Synthesized by Nippon Kayaku olfactory,epithelium ⁷) Co. only tested in cell-based models, not FDA-approved FD4AC3 Not tested Synthesized by Nippon Kayaku Co. only tested incell-based models, not FDA-approved FD5 AC5 (AC5 expressed in heart, Nottested Synthesized by Nippon Kayaku (NKH477) brain, kidney, liver, lung,uterus, Co. only tested in cell-based adrenal, brown adipose tissue ⁷)models, not FDA-approved FD6 AC5 Not tested Synthesized by Nippon KayakuCo. only tested in cell-based models, not FDA-approved

Abbreviations

ACTH: adrenocorticotropic hormone.AgRP: Agouti-related protein; a protein also produced in the arcuatenucleus and is an inverse agonist at MC4R. ProAgRP is processed to AgRPby PC1.cAMP: cyclic adenosine monophosphateGH: Ghrelin (the “hunger hormone”, also known as lenomorelin (INN), is apeptide hormone produced by enteroendocrine cells in the fundus of thestomach which functions as a neuropeptide in the central nervous system.proGHRH: progrowth hormone-releasing hormone.GHRH: Growth hormone-releasing hormone (GHRH), also known assomatoliberin or by several other names in its endogenous forms and assomatorelin (INN) in its pharmaceutical form, is a releasing hormone ofgrowth hormone (GH). It is a 44-amino acid peptide hormone produced inthe arcuate nucleus of the hypothalamus.PC1: Proprotein convertase 1, also known as prohormone convertase 1,prohormone convertase 3, proprotein convertase 3, neuroendocrineconvertase 1, or neuroendocrine convertase 3, and often abbreviated asPC1/3 is an enzyme that in humans is encoded by the PCSK1 gene. PCI andPC2, the protein products of the PCSK1 and PCSK2 genes, differentiallycleave many neuroendocrine or endocrine hormones, including,proopiomelanocortin, proinsulin, and proglucagon.PC2: Proprotein convertase 2 (PC2) also known as prohormone convertase 2or neuroendocrine convertase 2 (NEC2) is a serine protease andproprotein convertase PC2, like proprotein convertase 1 (PC1), is anenzyme responsible for the first step in the maturation of manyneuroendocrine peptides from their precursors, such as the conversion ofproinsulin to insulin intermediates. To generate the bioactive form ofinsulin (and many other peptides), a second step involving the removalof C-terminal basic residues is required; this step is mediated bycarboxypeptidases E and/or D. PC2 plays only a minor role in the firststep of insulin biosynthesis, but a greater role in the first step ofglucagon biosynthesis compared to PC1. PC2 binds to the neuroendocrineprotein named 7B2, and if this protein is not present, proPC2 cannotbecome enzymatically active. 7B2 accomplishes this by preventing theaggregation of proPC2 to inactivatable forms. The C-terminal domain of7B2 also inhibits PC2 activity until it is cleaved into smaller inactiveforms. Thus, 7B2 is both an activator and an inhibitor of PC2. Inhumans, proprotein convertase 2 is encoded by the PCSK2 gene. It isrelated to the bacterial enzyme subtilisin, and altogether there are 9different subtilisin-like genes in mammals: furin, PACE4, PC4, PC5/6,PC7/8, PCSK9, and SKI1/S1P.PCSK1: the gene encoding PC1.PCSK2: the gene encoding PC2.POMC: Pro-opiomelanocortin (POMC) is a precursor polypeptide with 241amino acid residues. POMC is synthesized in the pituitary from the285-amino-acid-long polypeptide precursor pre-pro-opiomelanocortin(pre-POMC), by the removal of a 44-amino-acid-long signal peptidesequence during translation.PDE4: phosphodiesterase 4.

PWS: Prader Willi Syndrome.

SNORD 116: SNORD116 (also known as HBII-85) is a non-coding RNA (ncRNA)molecule which functions in the modification of other small nuclear RNAs(snRNAs). This type of modifying RNA is usually located in the nucleolusof the eukaryotic cell which is a major site of snRNA biogenesis. It isknown as a small nucleolar RNA (snoRNA) and also often referred to as aguide RNA. SNORD116 belongs to the C/D box class of snoRNAs whichcontain the conserved sequence motifs known as the C box (UGAUGA) andthe D box (CUGA). Most of the members of the box C/D family function indirecting site-specific 2′-O-methylation of substrate RNAs. In the humangenome, there are 29 tandemly repeated copies of SNORD116, in the PWSregion of chromosome 15. In addition, other non-coding RNA species areendocing from the SNORD116 locus, including the long noncoding RNA,116HG, five sno-lncRNAs, and two spa-lncRNAs. SNORD116 is an orphannon-coding RNA locus that lacks clearly defined targets. Mouse modelslacking paternal Snord116 show similar symptoms to human PWS includinghyperphagia and growth deficiency.DPI devices/inhalers: dry powder inhalers; typically hand-held.MDI devices: metered-dose inhalers; typically hand-held.αMSH: is an endogenous ligand of the melanocortin 4 receptor.MC2R: melanocortin 2 receptor.MC4R: melanocortin 4 receptor.WT: wildtype.

Definitions

The term “pharmaceutically acceptable carrier”, as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent. Thediluent or carrier ingredients should not be such as to diminish thetherapeutic effects of the active compound(s).

The term “composition” as used herein means a product which results fromthe mixing or combining of more than one element or ingredient.

“Treating” or “treatment” of a state, disorder or condition includes:

(1) preventing or delaying the appearance of clinical symptoms of thestate, disorder, or condition developing in a person who may beafflicted with or predisposed to the state, disorder or condition butdoes not yet experience or display clinical symptoms of the state,disorder or condition; or

(2) inhibiting the state, disorder or condition, i.e., arresting,reducing or delaying the development of the disease or a relapse thereof(in case of maintenance treatment) or at least one clinical symptom,sign, or test, thereof; or

(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms or signs.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

“Patient” or “subject” refers to mammals and includes human andveterinary subjects.

“Inhibitors” and “antagonists,” or “activators” and “agonists,” refer toinhibitory or activating molecules, respectively, e.g., for theactivation of, e.g., a ligand, receptor, cofactor, a gene, cell, tissue,or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell,is a molecule that alters an activity of the gene, receptor, ligand, orcell, where activity can be activated, inhibited, or altered in itsregulatory properties. The modulator may act alone, or it may use acofactor, e.g., a protein, metal ion, or small molecule. Inhibitors arecompounds that decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate, e.g., a gene, protein, ligand, receptor,or cell. Activators are compounds that increase, activate, facilitate,enhance activation, sensitize, or up regulate, e.g., a gene, protein,ligand, receptor, or cell. An inhibitor may also be defined as acompound that reduces, blocks, or inactivates a constitutive activity.An “agonist” is a compound that interacts with a target to cause orpromote an increase in the activation of the target. An “antagonist” isa compound that opposes the actions of an agonist. An antagonistprevents, reduces, inhibits, or neutralizes the activity of an agonist.An antagonist can also prevent, inhibit, or reduce constitutive activityof a target, e.g., a target receptor, even where there is no identifiedagonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples, i.e., samples nottreated with antagonist, are assigned a relative activity value of 100%.Inhibition is achieved when the activity value relative to the controlis about 90% or less, typically 85% or less, more typically 80% or less,most typically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%. Activation is achieved when theactivity value relative to the control is about 110%, generally at least120%, more generally at least 140%, more generally at least 160%, oftenat least 180%, more often at least 2-fold, most often at least 2.5-fold,usually at least 5-fold, more usually at least 10-fold, preferably atleast 20-fold, more preferably at least 40-fold, and most preferablyover 40-fold higher.

The dosage of the therapeutic formulation will vary widely, dependingupon the nature of the disease, the patient's medical history, thefrequency of administration, the manner of administration, the clearanceof the agent from the host, and the like. The initial dose may belarger, followed by smaller maintenance doses. The dose may beadministered as infrequently as weekly or biweekly, or fractionated intosmaller doses and administered daily, semi-weekly, etc., to maintain aneffective dosage level. In some cases, oral administration will requirea higher dose than if administered intravenously. In some cases, topicaladministration will include application several times a day, as needed,for a number of days or weeks in order to provide an effective topicaldose.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, olive oil, sesame oil and the like. Water oraqueous solution saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as carriers, particularly forinjectable solutions. Alternatively, the carrier can be a solid dosageform carrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

Therapeutic compositions described herein may be administered by anymethod known in the art, including, without limitation, intranasal,oral, transdermal, ocular, intraperitoneal, inhalation, intravenous,ICV, intracisternal injection or infusion, subcutaneous, implant,vaginal, sublingual, urethral (e.g., urethral suppository),subcutaneous, intramuscular, intravenous, rectal, sub-lingual, mucosal,ophthalmic, spinal, intrathecal, intra-articular, intra-arterial,sub-arachinoid, bronchial or lymphatic administration. Topicalformulation may be in the form of gel, ointment, cream, aerosol, etc.;intranasal formulation can be delivered as a spray or in a drop;transdermal formulation may be administered via a transdermal patch oriontorphoresis; or, inhalation formulations can be delivered using anebulizer or similar device. Compositions can also take the form oftablets, pills, capsules, semisolids, powders, sustained releaseformulations, solutions, suspensions, elixirs, aerosols, or any otherappropriate compositions.

To prepare such pharmaceutical compositions, one or more PDE4 inhibitorsand/or one or more adenylate cyclase activators, and/or one or more MC4Ragonists may be mixed together with a pharmaceutical acceptable carrier,adjuvant and/or excipient, according to conventional pharmaceuticalcompounding techniques. Pharmaceutically acceptable carriers that can beused in the present compositions encompass any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, and emulsions, such as an oil/water or water/oil emulsion, andvarious types of wetting agents. The compositions can additionallycontain solid pharmaceutical excipients such as starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Liquid carriers, particularly for injectable solutions,include water, saline, aqueous dextrose, and glycols. For examples ofcarriers, stabilizers and adjuvants, see Remington's PharmaceuticalSciences, edited by E. W. Martin (Mack Publishing Company, 18th ed.,1990). The compositions also can include stabilizers and preservatives.

As used herein, the term a “therapeutically effective amount” is anamount sufficient to treat a specified disorder or disease oralternatively to obtain a pharmacological response treating a disorderor disease in vitro or in vivo in a mammal such as a human or non-humanpatient. Methods of determining the most effective means and dosage ofadministration can vary with the composition used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Treatment dosages generally may be titrated to optimizesafety and efficacy. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents can be readily determined by those of skill in the art. Forexample, the composition is administered at about 0.01 mg/kg to about200 mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg toabout 50 mg/kg. When the compounds described herein are co-administeredwith another agent or therapy, the effective amount may be less than,equal to or greater than when either agent is used alone.

Transdermal formulations may be prepared by incorporating the activeagent in a thixotropic or gelatinous carrier such as a cellulosicmedium, e.g., methyl cellulose or hydroxyethyl cellulose, with theresulting formulation then being packed in a transdermal device adaptedto be secured in dermal contact with the skin of a wearer. If thecomposition is in the form of a gel, the composition may be rubbed ontoa membrane of the patient, for example, the skin, preferably intact,clean, and dry skin, of the shoulder or upper arm and or the uppertorso, and maintained thereon for a period of time sufficient fordelivery of the PDE4 inhibitor and/or the adenylate cyclase activator tothe blood serum of the patient. The composition of the present inventionin gel form may be contained in a tube, a sachet, or a metered pump.Such a tube or sachet may contain one unit dose, or more than one unitdose, of the composition. A metered pump may be capable of dispensingone metered dose of the composition.

This invention also provides the compositions as described above forintranasal administration. As such, the compositions can furthercomprise a permeation enhancer. Southall et al. Developments in NasalDrug Delivery, 2000. The PDE4 inhibitor and/or the adenylate cyclaseactivator may be administered intranasally in a liquid form such as asolution, an emulsion, a suspension, drops, or in a solid form such as apowder, gel, or ointment. Devices to deliver intranasal medications arewell known in the art. Nasal drug delivery can be carried out usingdevices including, but not limited to, intranasal inhalers, intranasalspray devices, atomizers, nasal spray bottles, unit dose containers,pumps, droppers, squeeze bottles, nebulizers, metered dose inhalers(MDI), pressurized dose inhalers, insufflators, and bi-directionaldevices. The nasal delivery device can be metered to administer anaccurate effective dosage amount to the nasal cavity. The nasal deliverydevice can be for single unit delivery or multiple unit delivery. In aspecific example, the ViaNase Electronic Atomizer from Kurve Technology(Bethell, Wash.) can be used in this invention(http://www.kurvetech.com). The compounds of the present invention mayalso be delivered through a tube, a catheter, a syringe, a packtail, apledget, a nasal tampon or by submucosal infusion. U.S. PatentPublication Nos. 20090326275, 20090291894, 20090281522 and 20090317377.

The PDE4 inhibitor and/or the adenylate cyclase activator can beformulated as aerosols using standard procedures. The PDE4 inhibitorand/or the adenylate cyclase activator may be formulated with or withoutsolvents, and formulated with or without carriers. The formulation maybe a solution, or may be an aqueous emulsion with one or moresurfactants. For example, an aerosol spray may be generated frompressurized container with a suitable propellant such as,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen,carbon dioxide, or other suitable gas. The dosage unit can be determinedby providing a valve to deliver a metered amount. Pump spray dispenserscan dispense a metered dose or a dose having a specific particle ordroplet size. As used herein, the term “aerosol” refers to a suspensionof fine solid particles or liquid solution droplets in a gas.Specifically, aerosol includes a gas-borne suspension of droplets of aPDE4 inhibitor and/or the adenylate cyclase activator as may be producedin any suitable device, such as an MDI, a nebulizer, or a mist sprayer.Aerosol also includes a dry powder composition of the composition of theinstant invention suspended in air or other carrier gas. Gonda (1990)Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburnet al., (1992) Pharmacol. Toxicol. Methods 27:143-159.

The PDE4 inhibitor and/or the adenylate cyclase activator may bedelivered to the nasal cavity as a powder in a form such as microspheresdelivered by a nasal insufflator. The PDE4 inhibitor and/or theadenylate cyclase activator may be absorbed to a solid surface, forexample, a carrier. The powder or microspheres may be administered in adry, air-dispensable form. The powder or microspheres may be stored in acontainer of the insufflator. Alternatively, the powder or microspheresmay be filled into a capsule, such as a gelatin capsule, or other singledose unit adapted for nasal administration.

The pharmaceutical composition can be delivered to the nasal cavity bydirect placement of the composition in the nasal cavity, for example, inthe form of a gel, an ointment, a nasal emulsion, a lotion, a cream, anasal tampon, a dropper, or a bioadhesive strip. In certain embodiments,it can be desirable to prolong the residence time of the pharmaceuticalcomposition in the nasal cavity, for example, to enhance absorption.Thus, the pharmaceutical composition can optionally be formulated with abioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highlypurified cationic polysaccharide), pectin (or any carbohydrate thatthickens like a gel or emulsifies when applied to nasal mucosa), amicrosphere (e.g., starch, albumin, dextran, cyclodextrin), gelatin, aliposome, carbamer, polyvinyl alcohol, alginate, acacia, chitosansand/or cellulose (e.g., methyl or propyl; hydroxyl or carboxy;carboxymethyl or hydroxylpropyl).

The composition containing the PDE4 inhibitor and/or the adenylatecyclase activator can be administered by oral inhalation into therespiratory tract, i.e., the lungs.

Typical delivery systems for inhalable agents include nebulizerinhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI).

Nebulizer devices produce a stream of high velocity air that causes atherapeutic agent in the form of liquid to spray as a mist. Thetherapeutic agent is formulated in a liquid form such as a solution or asuspension of particles of suitable size. In one embodiment, theparticles are micronized. The term “micronized” is defined as havingabout 90% or more of the particles with a diameter of less than about 10μm. Suitable nebulizer devices are provided commercially, for example,by PARI GmbH (Starnberg, Germany). Other nebulizer devices includeRespimat (Boehringer Ingelheim) and those disclosed in, for example,U.S. Pat. Nos. 7,568,480 and 6,123,068, and WO 97/12687. The PDE4inhibitor and/or the adenylate cyclase activator can be formulated foruse in a nebulizer device as an aqueous solution or as a liquidsuspension.

DPI devices typically administer a therapeutic agent in the form of afree flowing powder that can be dispersed in a patient's air-streamduring inspiration. DPI devices which use an external energy source mayalso be used in the present invention. In order to achieve a freeflowing powder, the therapeutic agent can be formulated with a suitableexcipient (e.g., lactose). A dry powder formulation can be made, forexample, by combining dry lactose having a particle size between about 1μm and 100 μm with micronized particles of the present compounds and dryblending. Alternatively, the present compounds can be formulated withoutexcipients. The formulation is loaded into a dry powder dispenser, orinto inhalation cartridges or capsules for use with a dry powderdelivery device. Examples of DPI devices provided commercially includeDiskhaler (GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g.,U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat.No. 6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g.,U.S. Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g.,U.S. Pat. No. 4,353,365). Further examples of suitable DPI devices aredescribed in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 andreferences therein.

MDI devices typically discharge a measured amount of therapeutic agentusing compressed propellant gas. Formulations for MDI administrationinclude a solution or suspension of active ingredient in a liquefiedpropellant. Examples of propellants include hydrofluoroalklanes (HFA),such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227), and chlorofluorocarbons,such as CCl₃F. Additional components of HFA formulations for MDIadministration include co-solvents, such as ethanol, pentane, water; andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin. (See, for example, U.S. Pat. No. 5,225,183, EP 0717987, and WO92/22286). The formulation is loaded into an aerosol canister, whichforms a portion of an MDI device. Examples of MDI devices developedspecifically for use with HFA propellants are provided in U.S. Pat. Nos.6,006,745 and 6,143,227. For examples of processes of preparing suitableformulations and devices suitable for inhalation dosing see U.S. Pat.Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO 99/53901, WO00/61108, WO 99/55319 and WO 00/30614.

The PDE4 inhibitor may be encapsulated in liposomes or microcapsules fordelivery via inhalation. A liposome is a vesicle composed of a lipidbilayer membrane and an aqueous interior. The lipid membrane may be madeof phospholipids, examples of which include phosphatidylcholine such aslecithin and lysolecithin; acidic phospholipids such asphosphatidylserine and phosphatidylglycerol; and sphingophospholipidssuch as phosphatidylethanolamine and sphingomyelin. Alternatively,cholesterol may be added. A microcapsule is a particle coated with acoating material. For example, the coating material may consist of amixture of a film-forming polymer, a hydrophobic plasticizer, a surfaceactivating agent or/and a lubricant nitrogen-containing polymer. U.S.Pat. Nos. 6,313,176 and 7,563,768.

The PDE4 inhibitor and/or the adenylate cyclase activator can be givenalone or in combination with other drugs for the treatment of the abovediseases for a short or prolonged period of time, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50 or 60 days or 1, 2, 4, 5, 6, 7, 8, 9, 10,11 or 12 months or continuously over the lifetime of the patient. Thepresent compositions can be administered to a mammal, preferably a humanpatient. Mammals include, but are not limited to, mice, rats, rabbit,simians, bovines, ovine, porcine, canines, feline, farm animals, sportanimals, pets, equine, and primates.

The following are non-limiting examples.

Example 1

Manipulation of PC1 Expression and Activity in iPSC-Derived HypothalamicNeurons and β-Cells

Cyclase activation by Forskolin and/or inhibition of cAMP catabolism byinhibition of phosphodiesterase (Theophylline, IBMX) will increase PC1levels in in vitro models of PWS with consequential increases inprohormone processing. The identification of an apparent multi-tissuedeficiency in PCSK1 in in vivo and in vitro models of PWS enablesrational therapeutic targeting that may alleviate the majorneuroendocrine symptoms of PWS (FIG. 1).

The promoter region of the PCSK1 gene contains two cyclic adenosinemonophosphate (cAMP)-response elements (Conkright et al. 2003; Udupi etal. 1998). Agents that increase cellular levels of cAMP increase PCSK1mRNA and increase the secretion of prohormones processed by PC1 (FIG.2)(Udupi 1998). Forskolin and Theophylline are two FDA-approved drugswith generally safe treatment profiles in pediatric populations.Forskolin binds to adenylate cyclase close to its catalytic domainthrough hydrophobic interactions and hydrogen bonding (Tang and Hurley1998, Tesmer et al. 1999). Forskolin binding causes adenylate cyclaseconformation to change to its active form, thus increasing AC activityand increasing cellular cAMP levels (Onda et al 2001). Theophylline andother phosphodiesterase 4 (PDE4) inhibitors, such as MK0952, increasecellular cAMP levels by blocking its degradation.

Non-limiting examples of PDE4 inhibitors include Theophylline, MK0952,as well as the other PDE inhibitors in Tables 1A-B. Non-limitingexamples of adenylate cyclase (AC) activators include Forskolin and theactivators in Table 2.

Agents that can be used in the present method also include agents thatcan modify G protein activity, such as G protein activators orinhibitors, as well as G protein coupled receptor agonists.

NHLH2 and PCSK1 are downregulated in PWS microdeletion and largedeletion iPSC-derived neurons, as shown by RNA sequencing (FIGS. 3A,3C). FIG. 3A: NHLH2 is downregulated in PWS microdeletion and largedeletion iPSC-derived neurons compared to unaffected controls. FIG. 3B:NHLH2 protein is downregulated>90% in PWS microdeletion and largedeletion iPSC-derived neurons compared to unaffected controls. FIGS.3C-D: PCSK1 transcript and its protein product, PC1, aredownregulated>55% and >80%, respectively in PWS microdeletion and largedeletion iPSC-derived neurons compared to unaffected controls. FIGS. 3E-F: Mice in which only the paternal copy of Snord116 has been deleted(rest of PWS region is intact) display>40% downregulation of PC1 and PC2protein in isolated islets. FIG. 3G: Proinsulin is dependent on PC1 forits proper processing. There is a functional impairment in proinsulinprocessing in Snord116^(p−/m+) mice compared to WT littermates at 30minutes following glucose injection. FIG. 3H: There is 60% increase inthe ratio of proinsulin to insulin in the plasma of individuals with PWScompared to age and BMI-matched controls at fasting, indicating a defectin proinsulin to insulin processing. The effect is less than that seenin a patient with a PC1 mutation, consistent with the ˜50% reduction ofPC1 in PWS models. FIG. 3I: Proghrelin is also processed by PC1;proghrelin processing is impaired in stomach lysates fromSnord116^(p−/m+) mice compared to WT littermates. Stomach lysates fromPC1 null mice are included as a positive control for impaired proghrelinprocessing. FIG. 3J: ProGHRH processing may also be impaired inhypothalamic lysates from Snord116^(p−/m+) mice compared to WTlittermates, p=0.06. Impaired proGHRH processing is associated with lowcirculating GH and dwarfism in PC1 null mice. Snord116^(p−/m+) alsodisplay low GH and severe runting. FIG. 3K: Preliminary data suggeststhat treatment of unaffected control hypothalamic iPSC-derived neuronswith Forskolin (FSK) may increase transcript levels of PCSK1 in adose-dependent manner. POMC transcript levels may not be affected. FIG.3L: Treatment of unaffected control iPSC-derived β-cells with Forskolinincreases PCSK1 transcript levels. SNORD116 and INS transcript levelsmay be minimally affected. FIG. 3M: Transcript levels of Pcsk1 aredecreased 41% in Snord116p−/m+ hypothalamic at fasting; there is nodifference in Pcsk1 levels at refeeding. FIG. 3N: Transcript levels ofNhlh2 are decreased at both fasting and after refeeding in Snord116p−/m+hypothalamic compared to WT littermates. FIGS. 3O-P: Agrp and Npytranscript levels are increased in Snord116p−/m+ hypothalamic atrefeeding compared to WT. Follow-up, independent experiments confirmedthese changes by QPCR (gene expression) and Western blotting (proteinlevel) (FIGS. 3B, 3D). Individuals with PWS exhibit decreased fastinginsulin levels as compared to age and BMI matched controls. It washypothesized that this may be due to impaired proinsulin processing. Thepresent data illustrates that in a mouse model of PWS, in which only thepaternal copy of Snord116 is deleted, PC1 and PC2 protein levels aredecreased in isolated islets and are associated with a functionalimpairment in the processing of proinsulin to insulin (FIGS. 3E-3G).Proinsulin processing is also impaired (p=0.089) in plasma from humanPWS patients compared to age, BMI-matched controls at fasting (FIG. 3H).Plasma from a fasted patient harboring a PC1 mutation was included as apositive control for impaired proinsulin processing.

The hyperghrelinemia of PWS patients is a unique phenotype that may beassociated with impaired processing of proghrelin to mature ghrelin.Indeed, the present results illustrate that proghrelin to mature ghrelinprocessing was impaired in stomach lysates of Snord116^(p−/m+) micecompared to WT littermates (FIG. 3I). Stomach lysates from PC1 null micewere included as a positive control for impaired proghrelin processing.

Like individuals with PWS, patients with PCI mutation have decreasedcirculating GH levels. Mice null for PC1 have severe runting anddecreased circulating GH associated with impaired proGHRH to GHRHprocessing. We found that Snord116^(p−/m+) mice, which are also runtedand have low circulating GH, trend towards impaired processing ofproGHRH to GHRH in hypothalamic lysates (FIG. 3J).

As outlined in FIG. 2, the identification of a decrease in PC1 andimpaired prohormone processing in PWS suggests a unified moleculartheory which may account for many of the neuroendocrine features of thedisease. Thus, agents that increase PC1 activity and thereby increaseprohormone processing, represent a rational, targeted therapy for themajor neuroendocrine features of PWS.

Forskolin is known to increase cellular Pcsk1 levels and can increaseprohormone processing. Forskolin was applied to non-PWS iPSC-derivedneurons and β-cells and the results illustrate that PCSK1 transcriptlevels increased compared to untreated cells (FIGS. 3K-L). Studies inPWS-derived neurons and β-cells will be conducted.

The response of PCSK1 transcript levels, PC1 protein levels, andrelevant prohormone processing levels will be tested in in vitro and invivo model systems. We will treat unaffected control and hypothalamiciPSC-derived neurons with graded levels of Forskolin, Theophylline, andForskolin+Theophylline and measure PC1 transcript and protein, POMCtranscript and protein, as well as protein levels of processed productsof POMC including: αMSH, β-endorphin, and ACTH. These peptides will bequantitated in whole cell lysates as well as the levels secreted intothe cellular medium. The cells will be treated with differentconcentrations of Forskolin and Theophylline in order to determinewhether PC1 levels can be increased in a dose-dependent manner and inorder to identify an optimal dosage range to increase PC1 levels andPOMC processing. Other PDE4 and adenylate cyclase inhibitors will betested in these assays as well (see, Tables 1A-B and 2).

Batch RNA sequencing and/or single cell RNA sequencing will be performedto identify other transcripts that are most affected by thepharmacological treatments. This approach is expected to predictoff-target effects upon in vivo treatment. Single cell RNA sequencingwill be especially informative regarding PCSK1 transcript increasesfollowing treatment in POMC-expressing neurons. Other adenylate cyclaseactivators and PDE4 inhibitors (Tables 1A-B, 2) will be tested iniPSC-derived hypothalamic neurons following the same study protocol asdescribed above.

We will differentiate iPSC from unaffected control and PWS (large andminimum deletion) to iPSC-derived β-cells. We will treat theiPSC-derived β-cells with Forskolin, Theophylline, andForskolin+Theophylline and measure levels of PC1 at the transcript andprotein level. We will also measure INS transcript levels as well asprotein levels of proinsulin, insulin, and c-peptide from whole celllysates as well as the concentrations of the proteins secreted by theβ-cells into the media. These cells may be transplanted into nude miceto enable their maturation; these cells can be tested in vivo forinsulin processing; excised cells will be tested as described. We willalso use human isolated islets from non-diabetic, non-obese individuals(available to us through the National Pancreatic Donors Registry) totest the effects of Forskolin, Theophylline, and Forskolin+Theophyllineon PC1 levels in fully mature human pancreatic islets.

Example 2

Confirmatory molecular physiology of PC1 metabolism in Snord116^(p−/m+),Pc1^(−/−) and Pc1^(+/−) Mice.

Increasing cAMP levels by adenylate cyclase (AC) activation andconcurrent PDE inhibition will increase PC1 levels in ex vivo and invivo models of PWS, and may consequently increase prohormone processingin the Snord116^(p−/m+) mouse model of PWS. Because mice with a paternaldeletion of Snord116 (a mouse model of PWS) have impaired processing ofproinsulin to insulin associated with reductions in PC1 transcript andprotein, islets will be isolated from wild type (WT) andSnord116^(p−/m+) mice and the responses of these cells to Forskolin,Theophylline, and Forskolin+Theophylline will be analyzed. The samemanipulations and measurements will be performed as for iPSC-derivedβ-cells. If proinsulin processing can be rescued in isolated islets fromSnord116^(p−/m+) mice as compared to WT littermates, then investigationsof proinsulin processing rescue in Snord116^(p−/m+) mice treated withForskolin, Theophylline, and Forskolin+Theophylline, in vivo will beconducted.

The present results illustrate that Snord116^(p−/m+) mice havereductions in proinsulin processing to insulin that are associated withreduced PCI and PC2 content in the islets (FIGS. 3E-G). Furthermore,proghrelin processing is also impaired in the stomachs ofSnord116^(p−/m+) mice as well as the processing of proGHRH to GHRH inthe hypothalami of Snord116^(p−/m+) mice compared to WT littermates(FIGS. 3I-J). Furthermore, the ratio of proinsulin to insulin iselevated in fasted individuals with PWS compared to age and BMI matchedcontrols, suggesting an impairment in the processing of proinsulin toinsulin (FIG. 3H).

Isolated islets from Pa null and heterozygous mice will be included as acontrol for impaired proinsulin processing as well as a predictednegative response to pharmacological treatment. Peripheral levels ofglucose, proinsulin, insulin, and c-peptide will be measured at fasting,and 15, 30, 60, and 120 minutes following intraperitoneal glucoseinjection. Optimal duration of pharmacological treatment inSnord116^(p−/m+) and WT mice prior to peripheral measures of proinsulinprocessing will be established empirically. Pa null and heterozygousmice will be included as a control for impaired proinsulin processing inin vivo experiments as well. The initial time periods for testing willbe 3 days, 1 week, and 1 month. Several methods of drug delivery willalso be tested.

Assays that can distinguish between proghrelin and mature ghrelin, andcan thus be used to measure proghrelin processing in the circulationwill be developed for both human and mouse. Proghrelin processingfollowing the in vivo pharmacological treatments described above inSnord116^(p−/m+), PC1 null, PC1 heterozygous, and WT animals will bemeasured.

The hypothalami of Snord116^(p−/m+), WT, and Pa null and Pa heterozygousmice treated with Forskolin, Theophylline, and Forskolin+Theophylline,and measure protein levels of PC1, POMC, αMSH, β-endorphin, and ACTHwill be analyzed in order to assess whether in vivo treatment can affectlevels of PCI and POMC processing.

Because Snord116^(p−/m+) animals are runted and do not develop obesity,the main model by which POMC processing will be assessed is theiPSC-derived human neurons, in which more extreme downregulations ofNHLH2 and PC1 are observed. However, PC1 and POMC responses to thesepharmacologic agents may also be analyzed in primary neurons from youngWT POMC-GFP mice. POMC-expressing neurons can be specifically isolatedusing mice in which POMC neurons express GFP. We will knock down Pcsk1as a control for impaired POMC processing. We may also try to knock downspecific isoforms of Snord116 in vitro using siRNA- or2-O-methyl-modified anti-sense oligo-based approaches; siRNA are small,double-stranded interfering RNAs that are commonly used to knock downcytoplasmic RNAs, while 2-O-methyl-modified anti-sense oligos are usedto knock down snoRNAs which typically are found in the nucleolus (Lianget al. 2011). We will then measure PC1 levels and POMC processing levelsand investigate whether pharmacological treatment can increase levels ofPC1 and increase POMC processing in the primary mouse neurons in whichSnord116 has been knocked down relative to WT.

Additionally, mice with conditional hypomorphic alleles of SNORD116 willbe obtained or created. Adult animals with such alleles will haveSnord116 acutely reduced in specific hypothalamic nuclei (e.g. thearcuate nucleus) by introduction of suitable cre-expressing constructs,including those driven by specific promoters, eg. For POMC. Thisapproach will circumvent the somatic developmental effects (stunting) ofSnord116 hypomorphism in mice.

Effect of cyclase activator and/or phosphodiesterase inhibitor=50% orgreater increase in relevant prohormone processing in at least one modeltested: PWS iPSC-derived neurons, PWS iPSC-derived β-cells,Snord116^(p−/m+) isolated islets, Snord116^(p−/m+) circulatingprohormones, or Snord116-knockdown primary neurons. These phenotypeswould be accompanied by increases in relevant transcripts and/orproteins in the affected cells.

Cyclase Activation

Forskolin was applied to various cellular models and the resultsillustrate that it robustly and reliably increases PCSK1 transcriptlevels, PC1 protein levels, and functionally increases prohormoneprocessing. PCSK1/Pcsk1 transcript levels increased between 2-3 fold iniPSC-derived neurons and primary mouse neurons exposed to 10 μMForskolin (FIGS. 5A, C, D).

Cyclic AMP concentrations increased about 8.5-fold in iPSC-derivedhypothalamic ARC neurons exposed to 10 μM Forskolin, supporting theinference that application of Forskolin increases PCSK1 transcriptlevels by raising cellular cAMP levels which in turn activate PCSK1'scAMP-response element promoter. FIG. 5A is a graph showing primaryforebrain neurons were isolated from gestational day 19.5 (E19.5)embryos of wild type mice and cultured for 72 hours. Subsequently, cellswere exposed to either 10 μM Forskolin or its vehicle, dimethylsulfoxide (DMSO) for 20 hours. Pcsk1 transcript increased ˜2.5-fold inprimary neurons exposed to Forskolin. FIG. 5B is a graph showingunaffected control hypothalamic arcuate-like (ARC) neurons (Hes Nkx2-1hESC line) at day 37 (D37) of differentiation were treated with 10 μMForskolin or vehicle for thirty minutes. Cyclic adenosine monophosphate(cAMP) levels were increased about 8.5-fold in cells exposed toForskolin. FIG. 5C is a graph showing exposure of unaffected controlhypothalamic iPSC-derived neurons (line 1023A) at day 30 ofdifferentiation to graded concentrations of Forskolin elicits adose-dependent response in PCSK1 transcript levels. FIG. 5D is a graphshowing exposure of iPSC-derived neurons (line 1043D3) to 10 μMForskolin for multiple time intervals finds that PCSK1 is notsignificantly upregulated after only 1 hour of exposure, but issignificantly upregulated by 4 and 18 hours of exposure. Four hours ofexposure yielded the maximal increase in upregulation, about 2.5-fold,of the time points tested. FIGS. 5E-F are graphs showing treatment ofunaffected control (1023A) iPSC-derived hypothalamic ARC neurons at day30 of differentiation with graded concentration of Forskolin identifiesa dose-dependent increase in POMC processing to both β-endorphin (βEP)and α-melanocyte stimulating hormone (αMSH). FIGS. 5G-H are graphsshowing treatment of isolated islets from adult (8-12 week old) WT micewith multiple concentrations of Forskolin shows upregulation of PC1, butnot PC2, protein levels at 25 and 50 μM Forskolin concentrations,respectively.

Treatment with forskolin not only elevated transcript levels but alsohad functional consequences in that POMC processing to both β-endorphinand αMSH were increased (FIGS. 5E-F). Furthermore, application ofForskolin to isolated islets from wild type mice resulted in ˜3-foldincrease in PC1 protein levels (FIG. 5 G). PC2 protein levels wereunaffected (FIG. 5H). Taken together, these results show that PC1 levelsincrease in response to Forskolin in three separate model systems:iPSC-derived neurons, primary neurons, and isolated islets. Furthermore,Forskolin-induced elevation of PC1 is functionally consequential,resulting in increased levels of prohormone processing.

Phosphodiesterase Inhibition

PDE inhibitors have also been tested in iPSC-derived neurons and foundthat inhibition of phosphodiesterase can also increase transcription ofPCSK1. However, the effect size of PDE inhibition on PCSK1 transcriptlevels is less than that induced by AC agonism with forskolin.Theophylline (10 mM) and Roflumilast (1 mM) both increase PCSK1transcription as single agents, while MK0952 has thus far only beenfound to increase PCSK1 transcription in combination with Forskolin invitro (FIGS. 6A-C, F). Combination treatment with Forskolin andRoflumilast demonstrates that these agents can work together in anadditive, possibly synergistic, manner inducing and increased PCSK1transcription at lower concentrations (1 μM Forskolin, 100 nMRoflumilast) than when either agent is given alone (FIG. 6D). Again,increased PCSK1 transcription due to combination treatment withForskolin and Roflumilast also increases prohormone processing of POMCto ACTH (FIG. 6E). Specifically, tests with graded concentrations ofForskolin in isolated mouse islets showed a 3-fold upregulation of PC1protein at 25 μM and 50 μM concentrations. No change in PC2 proteinlevels were observed in response to Forskolin application. We also foundthat 10 μM Forskolin applied to primary mouse neurons isolated fromE19.5 mice increased Pcsk1 transcript levels ˜2-fold.

MK0952

The phenotype most limiting to PWS patients is hyperphagia which is mostlikely mediated by processes occurring in the central nervous system,particularly the hypothalamus. Thus agents that aim to amelioratehyperphagia must be able to penetrate the blood brain barrier. MK0952 isan intrinsically potent (IC₅₀=0.6 nM) brain-penetrant PDE4 inhibitorwith limited whole blood activity (IC₅₀=555 nM) (M. Gallant et al.2010). As described herein, MK0952 is the lead PDE inhibition candidateat present.

A preliminary in vivo test of MK0952 was performed in wild type mice. Asingle administration of MK0952 at 10 mg/kg body weight results in a 25%increase in hypothalamic Pcsk1 transcript levels (FIG. 7A).Administration of Forskolin at 25 mg/kg did not result in increasedhypothalamic Pcsk1 transcript levels. Co-administration of MK0952 at 10mg/kg and Forskolin at 25 mg/kg again induced an ˜25% increase inhypothalamic Pcsk1 levels, suggesting that this increase was dueprimarily to the actions of MK0952 (FIG. 7B). We will also analyzecirculating cAMP levels, cortical proBDNF/BDNF, cerebellar Pcsk1,gastric Pcsk1, and gastric proghrelin/ghrelin, circulating proinsulinand insulin concentrations (and their ratios), and finally pulmonaryPcsk1 transcript and cAMP levels from these animals as well.

The first in vivo study with MK0952 administered by oral gavage whileforskolin was administered intraperitoneally one time to ˜4 hour fastedwild type mice was completed. Hypothalamic transcript levels of Pcsk1were upregulated ˜25% following administration of either 10 mg/kg MK0952as a single agent or both 10 mg/kg MK0952 and 25 mg/kg Forskolin.However, administration of 25 mg/kg Forskolin only did not result in anupregulation of hypothalamic Pcsk1, suggesting the Forskolin at thisdose does cannot access the hypothalamus in sufficient quantities toaffect Pcsk1 transcription. This also suggests that the increase inhypothalamic Pcsk1 following administration of both 25 mg/kg Forskolinand 10 mg/kg MK0952 was mainly due to the actions of MK0952 in thehypothalamus. This difference likely reflects greater CNS penetrance ofthe MK0952, not the general relevance of a cyclase activator to therapyof PWS. No change in the ratio of circulating proinsulin:insulin wasdetected following administration MK0952 or Forskolin. Because theprocessing of proinsulin to insulin is already quite efficient in WTanimals, it is in hindsight unlikely that it would further increase atfasting. However, these ‘baseline’ data are still valuable for assessingproinsulin to insulin processing under the setting of an intraperitonealglucose tolerance test at 3 mg/kg glucose in both WT andSnord116^(p−/m+) mice. Additionally, samples were collected formeasurement of circulating cAMP levels, cortical proBDNF/BDNF,cerebellar Pcsk1, gastric Pcsk1, and gastric proghrelin/ghrelin, andfinally pulmonary Pcsk1 transcript and cAMP levels.

Example 3

Clinical Study of Compounds in Patients with Prader Willi Syndrome (PWS)

The preliminary design of the proposed clinical study for individualswith PWS is based on the hypothesis that the expression of proconvertase1 (PC1) in decreased in the neurons of individuals with PWS (Burnett etal. 2017). Experimental in vitro and in vivo exposure to adenylatecyclase agonists and PDE4 inhibitors causes up-regulation of PC1expression and activity in human stem cell-derived and rodent forebrainneurons, and human fibroblasts. It is anticipated that administration ofthese drugs will increase the conversion of implicated prohormones toactive hormones.

The clinical study will address and illustrate the efficacy of the drugin enhancing the activity of PC1 as follows:

-   -   1. To illustrate the effects of the candidate therapeutic agents        on the behavioral and the endocrine phenotypes of PWS.    -   2. To monitor the clinical safety profile of such agents.

Study Design:

The clinical study will utilize a cross-over study design (Cleophas etal. 2006, Wellek and Blettner 2012, and Louis et al. 1984). This designprovides the power to assess the effect of treatment accounting for thevariability between subjects in a small cohort (FIG. 8A). The washoutperiod between the two treatment arms will mitigate carryover effects;the short duration of the study minimizes the “time-effects” (effects onthe change in disease process over time).

Inclusion Criteria*:

-   -   1. Genetically proven diagnosis of PWS    -   2. Age>18 years        -   *Recombinant growth hormone therapy is permissible.

Exclusion Criteria:

-   -   1. Severe psychiatric disorder    -   2. Uncooperative to take the medication    -   3. Systemic illness, e.g. serious gastrointestinal illness like        inflammatory bowel disease, cardiac disease, especially rhythm        disturbances, diagnosis of diabetes, hepatic or renal disease or        failure.    -   4. Anemia defined as hemoglobin<10 gm/dL    -   5. Patients on drugs that have potential interaction with the        target drug, e.g. PDE4 inhibitors interact with anti-seizure        medications, cimetidine, omeprazole, antibiotics etc. Many of        these drugs alter hepatic enzyme activity and could interfere        with the metabolism of a PDE4 inhibitor. A complete list of        exclusion drugs will be based on the pharmacokinetic and        pharmacodynamic properties of the identified therapeutic agent.

Recruitment: The recruitment of the subjects will be facilitated bypartnership with the PWS Foundation (FPWR and PWSA), patient supportgroups and the clinicians caring for children with PWS. Phone screeningwill identify potentially eligible subjects who will be invited for thescreening visit.

The study will last for 4-6 weeks and consist of the following visits:

-   -   1. Screening visit: At this visit, a complete review of medical        records, medications and physical examination will be performed        along with screening lab measurements (from FIG. 8B, same as        safety profile aside from the drug level). Subjects will be        provided a week of placebo for the run-in period to assess        compliance. This will be a short outpatient visit (˜3 hours).        All other study visits will be 6-8 hours long short in-patient        stay.    -   2. Baseline visit (t1 and t3 from FIG. 8A): Subjects who        successfully complete the run-in period will be invited to        participate in the study. The eligible subjects will be        randomized to either the AP group, or the PA group (FIG. 8A).        Subjects will be advised to fast for >8 hours for the visit.        Physical profiling will include height, weight, body fat        measurement, vital signs, resting energy expenditure and a        complete physical examination. A complete pituitary profile that        includes ACTH, cortisol, FSH/LH, estrogen/testosterone, TSH/free        T4, GH, IGF-1, IGFBP3 will be performed. The subjects will        undergo a mixed meal tolerance test (MMTT) with standardized        meal and blood measurements will be obtained at 0,30,60,90,120        and 180 minutes from an indwelling IV catheter (FIG. 8B).

The primary guardians will complete questionnaires relating tohyperphagia (Dykens or modified Dykens) and behavioral assessment willbe performed using the Oxytocin Study Questionnaire (25-28). In additionto this, they will complete a food frequency questionnaire on 3 separatedays to include at least one weekend. The visit is expected to last 6-8hours. Study medication for 1 week will be dispensed with caregiverinstruction.

-   -   3. Follow-up visit (t2 and t4 from FIG. 1): The subject will        return for a follow-up visit in 1 week after the initiation of        the study medication. The measurements mentioned above will be        repeated at this visit, and a medication count will be obtained,        along with structured questionnaire for the assessment of        toxicity. Each of these visits will be 6-8 hours. A washout        period of 1-2 weeks will be allowed prior to the 2^(nd) phase of        the study.

Outcome Measures:

-   1. Hormonal profile in response to a standard meal: Based on the    effect of PCI on conversion of prohormones (such as proinsulin to    insulin etc.), it is anticipated that administration of the drug    will cause an increase in the ratio of prohormone: hormone (e.g.    proinsulin: insulin) (Burnett et al. 2017). This will be tested by    hormonal response to a standard MMTT. MMTT is performed by    administration of a liquid meal (6 cc/kg of Boost or equivalent to a    maximum of 360 cc) followed by periodic measurement of insulin,    proinsulin, POMC prohormone, ACTH, AgRP, proglucagon, glucagon,    GLP1, oxytocin (and propeptide), ghrelin, proghrelin, free fatty    acids, and glucose. MMTT has been validated in clinical studies of    subjects with PWS (P. Gumus Balikcioglu et al. 2015).

Relative to values obtained prior to drug administration, it isanticipated that there will be an absolute increase in insulin release,decrease in proinsulin release and an increase in insulin/proinsulinratio. All in 25% range. It is also expected that glucose concentrationswill be decreased by 15-20 and ffa as well. In plasma obtained prior tothe MMT, it is anticipated that POMC will be increased and AgRP reducedby ˜25%. Oxytocin should be increased by 15-20% as well. It is alsoanticipated that proghrelin/ghrelin ratio will be reduced. Spinal fluidmay also be examined/studied in these subjects as well, but would not beevaluated in relationship to a meal. The following components may beassayed: pomc prohormone, beta endorphin, alpha msh, AgRP and oxytocin,anticipating that the drugs would reduce pomc prohormone, and increasebeta endorphin, alpha msh and oxytocin, and reduce AgRP in comparison tountreated subjects.

-   2. Pharmacometabolomic profile: Metabolomic profiling provides an    additional opportunity to understand the effects of the drug on the    metabolic phenotype. Metabolomic profile of the study subjects    before and after treatment with the drug will be performed to    identify biomarkers for a) response to treatment in the pathways of    interest, viz. insulin metabolism pathway and others, b) to identify    individual differences in treatment, by identification of pathways    selectively up- or down-regulated in different individuals, and c)    identify the profiles of side-effects or toxicity using a pathway    based analysis that may not be obvious by the standard study of    established larger molecular profiling (R. Kaddurah-Daouk, R.    Weinshilboum, N. 2015; R. D. Beger et al. 2016).-   3. Changes in Hyperphagia related behavior: The Dykens (and modified    Dykens) questionnaire assesses the behavior, severity and drive for    hunger. In addition to these outcomes, the Oxytocin Behavior    Questionnaire will assess social and emotional behavior related with    eating. These outcomes will be supplemented with the analysis of the    food frequency questionnaires. It is expected that treatment will    also improve behavior and/or emotional state.

Sample size: A pilot sample size of 6 subjects will be recruited forthis study. The power of this sample size to detect outcomes of interestwill depend on the effect size ascertained by in vivo hormonal orpharmacometabolomic profiling in animal models. As reflected in FIG. 8C,an effect size of ˜1.47 is required to detect a significant change in acohort of 6 subjects. The effect size is calculated as the difference inthe means of the observation with the placebo as compared to the activedrug divided by the standard deviation of the change. As each subjectserves as his/her own control, the crossover study design limits thevariability and allows reaching power in ¼ of the subjects for a similarparallel arm study design.

Additional Study Information:

-   -   1. Based on the prior studies in children with PWS and the need        to achieve high effect size, the standard mixed meal has been        selected for the study.    -   2. The study will be conducted in the outpatient facility of the        Irving Institute for Clinical and Translational Research.    -   3. An IRB protocol for the study will be prepared directed to        the appropriate PDE inhibitor and/or cyclase activator.    -   4. The metabolomic profiling—if obtained in this preliminary        study—will be performed in the Hormone and Metabolite Core of        the Diabetes and Endocrinology Research Center.

Example 4 Methods of Treating Prader-Willi Syndrome—Combination Therapyof Endogenous and Exogenous MC4R Agonism.

Individuals with PWS will be treated using agents that increaseendogenous levels of processed hormones by virtue of increasing PC1production through raising levels of cellular cAMP production and/orblocking its degradation.

In the arcuate nucleus, POMC is processed to αMSH by proconvertase 1(PC1) (S. L. Wardlaw 2011). αMSH is an endogenous ligand of themelanocortin 4 receptor (MC4R). Humans and mice with inactivatingmutations in POMC, PCSK1 (gene product of PCSK1 is PC1), or MC4R arehyperphagic and obese (C. Vaisse et al. 1998). Mutations in MC4R are themost common single gene cause of obesity in humans (R. J. Loos et al.2008). AgRP is also produced in the arcuate nucleus and is an inverseagonist at MC4R. ProAgRP is processed to AgRP by PCI (S. L. Wardlaw2011).

It is possible that increases in PCI production may increase theproduction of both αMSH and AgRP, which have opposite effects at MC4R(FIG. 4). The use of small molecule or peptide-based MC4R agonists couldhelp to ensure that the extracellular pools of agents that agonize MC4Rare in excess of those that antagonize MC4R (FIG. 4, Table 3). Thiswould be expected to push signaling at the MC4R towards anorexigenicresponses (FIG. 4). Agents that bind to AgRP and block its effects atMC4R could also be a useful strategy in this setting (Table 3) (E. C.Lee and P. A Carpino 2016). Compounds that act similarly that are notmentioned in Table 3 may also be useful. This strategy may beefficacious not just for treating PWS, but also other forms ofmonogenic/syndromic obesity as well as common obesity.

TABLE 3 Example Compounds that could be co-administered with FSK and PDEInhibitors Mechanism of Drug Name Company Action Notes RM-493 RhythmPeptide-based Currently in Phase II (Setmelanotide) Pharmaceuticalsanalog of α-MSH clinical trials for treatment and Ipsen of hyperphagiain PWS, pharmaceuticals reported that does not have (Ipsen patentedoff-target cardiovascular slow-release effects, administration viaformula) subcutaneous injection TTP2515 Transtech Small molecule,Decreases food intake and Pharma non-peptide weight gain in lean miceAgRP₈₃₋₁₂₂ on high fat diet antagonist, does not block activity of αMSH2-aminothiazole Transtech Alternative derivatives Pharma formulations ofabove TTP2515 MK-0493 Merck Small molecule, Robust weight loss innon-peptide animal models but limited MC4R agonist efficacy for weightloss in non-genetic obese; possible that individuals with PWS may haveincreased sensitivity to such agents, no observed cardiovascular sideeffects, oral formulation

Summary/Conclusions

Although the gene encoding PC1, PCSK1, is downregulated in cell basedand animal models of PWS, the gene itself is intact and thus could besubject to pharmacological manipulation. The present data providesresults of ongoing preclinical studies to pharmacologically manipulatecellular levels of PCSK1/PC1. In vitro experiments demonstrate thatapplication of Forskolin, an adenylyl cyclase agonist robustly andreliably upregulates PCSK1 expression in human stem cell-derivedneurons, mouse primary neurons, and increases PC1 protein level in mouseisolated islets. Furthermore, Forskolin treatment also increases POMCprohormone processing in stem cell-derived hypothalamic neurons.Application of PDE inhibitors Theophylline and Roflumilast to stem cellneurons increases PCSK1 transcript levels both as single agents and incombination with Forskolin. Combination treatment of Roflumilast andForskolin also additively increases POMC prohormone processing (toanorexigenic peptides) in stem cell hypothalamic neurons. Treatment ofstem cell-derived neurons with both Forskolin and MK0952 (a class 4 PDEinhibitor) increases PCSK1 mRNA. Finally, a single oral dose of 10 mg/kgMK0952 increases hypothalamic Pcsk1 transcript levels by 25% in wildtype mice. Longer applications of MK0952 in vivo in both wild type andmice hypomorphic for paternal Snord116 will be tested next. In additionwe will collaborate with Andrea Haqq and colleagues to measurecirculating pro- and processed hormone levels (e.g. proinsulin, POMC,pro-oxytocin, proBDNF) in individuals with PWS and matched controls.

Also provided is a protocol for preliminary clinical study of MK0952 andother candidate compounds in individuals with PWS. The major aims ofthis clinical study will be to monitor the clinical safety profile ofthese agents in PWS subjects as well as to measure behavioral andneuroendocrine endpoints to assess preliminary efficacy.

REFERENCES

-   Conkright, M. D. et al. Genome-Wide Analysis of CREB Target Short    Article Genes Reveals A Core Promoter Requirement for cAMP    Responsiveness. Molecular Cell 11, 1101-1108 (2003).-   Udupi, V., Townsend, C. M. & Greeley, G. H. Stimulation of    Prohormone Convertase-1 mRNA Expression by Second Messenger    Signaling Systems. BIOCHEMICAL AND BIOPHYSICAL RESEARCH    COMMUNICATIONS 246, 463-465 (1998).-   Tang, W.-J. & Hurley, J. Catalytic Mechanism and Regulation of    Mammalian Adenylyl Cyclases. Molecular Pharmacology 54, 231-240    (1998).-   Tesmer, J. J. G. et al. Two-Metal-Ion Catalysis in Adenylyl Cyclase.    Science 285, 756-760 (1999).-   Onda, T. et al. Type-specific regulation of adenylyl cyclase.    Selective pharmacological stimulation and inhibition of adenylyl    cyclase isoforms. J Biol Chem 276, 47785-47793,    doi:10.1074/jbc.M107233200 (2001).-   Liang, X. H., Vickers, T. A., Guo, S. & Crooke, S. T. Efficient and    specific knockdown of small non-coding RNAs in mammalian cells and    in mice. Nucleic Acids Res 39, e13, doi:10.1093/nar/gkq1121 (2011).-   Sunahara, R. K. & Taussig, R. Isoforms of Mammalian Adenylyl    Cyclase: Multiplicities of Signaling. Molecular Interventions 2,    168-184 (2002).-   S. L. Wardlaw, Hypothalamic proopiomelanocortin processing and the    regulation of energy balance. European journal of pharmacology 660,    213-219 (2011).-   C. Vaisse, K. Clement, B. Guy-Grand, P. Froguel, A frameshift    mutation in human MC4R is associated with a dominant form of    obesity. Nature Genetics 20, 113-114 (1998).-   R. J. Loos et al., Common variants near MC4R are associated with fat    mass, weight and risk of obesity. Nat Genet 40, 768-775 (2008).-   E. C. Lee, P. A. Carpino, Melanocortin-4 receptor modulators for the    treatment of obesity: a patent analysis (2008-2014). Pharmaceutical    Patent Analyst 4, 95-107 (2016).-   Fimia G M, Sassone-Corsi P. 2001. Cyclic AMP signaling. J Cell Sci    114: 1971-1972.-   L. C. Burnett et al., Deficiency in prohormone convertase PC1    impairs prohormone processing in Prader-Willi syndrome. J Clin    Invest 127, 293-305 (2017).-   F. D. P. Deborah J. Good, Kathleen A. Mahon, Albert F. Parlow,    Heiner Westphal, Ilan R. Kirsch, Hypogonadism and obesity in mice    with a targeted deletion of the Nhlh2 gene. Nature Genetics 15,    397-401 (1997).-   D. L. Fox, Dissertation, University of Massachusetts Amherst, Ann    Arbor, Mich. (2007).-   P. Stijnen, B. Ramos-Molina, S. O'Rahily, J. W. M. Creemers, PCSK1    mutations and human endocrinopathies: from obesity to    gastrointestinal disorders. Endocrine Reviews 17, (2016).-   M. D. Conkright et al., Genome-Wide Analysis of CREB Target Short    Article Genes Reveals A Core Promoter Requirement for cAMP    Responsiveness. Molecular Cell 11, 1101-1108 (2003).-   V. Udupi, C. M. Townsend, G. H. Greeley, Stimulation of Prohormone    Convertase-1 mRNA Expression by Second Messenger Signaling Systems.    Biochemical and Biophysical Research Communications 246, 463-465    (1998).-   W.-J. Tang, J. Hurley, Catalytic Mechanism and Regulation of    Mammalian Adenylyl Cyclases. Molecular Pharmacology 54, 231-240    (1998).-   J. J. G. Tesmer et al., Two-Metal-Ion Catalysis in Adenylyl Cyclase.    Science 285, 756-760 (1999).-   T. Onda et al., Type-specific regulation of adenylyl cyclase.    Selective pharmacological stimulation and inhibition of adenylyl    cyclase isoforms. J Biol Chem 276, 47785-47793 (2001).-   M. Gallant et al., Discovery of MK-0952, a selective PDE4 inhibitor    for the treatment of long-term memory loss and mild cognitive    impairment. Bioorganic & Medicinal Chemistry Letters 20, 6387-6393    (2010).-   Q. Zhang, G. J. Bouma, K. McClellan, S. Tobet, Hypothalamic    expression of snoRNA Snord116 is consistent with a link to the    hyperphagia and obesity symptoms of Prader-Willi syndrome. Int J Dev    Neurosci 30, 479-485 (2012).-   Y. Qi et al., Snord116 is critical in the regulation of food intake    and body weight. Sci Rep 6, 18614 (2016).-   L. Wang et al., Differentiation of hypothalamic-like neurons from    human pluripotent stem cells. J Clin Invest 125, 796-808 (2015).-   V. Grinevich, M. G. Desarmenien, B. Chini, M. Tauber, F. Muscatelli,    Ontogenesis of oxytocin pathways in the mammalian brain: late    maturation and psychosocial disorders. Front Neuroanat 8, 164    (2014).-   M. Tauber et al., The Use of Oxytocin to Improve Feeding and Social    Skills in Infants With Prader-Willi Syndrome. Pediatrics 139,    (2017).-   R. J. Kuppens, S. H. Donze, A. C. Hokken-Koelega, Promising effects    of oxytocin on social and food-related behaviour in young children    with Prader-Willi syndrome: a randomized, double-blind, controlled    crossover trial. Clin Endocrinol (Oxf) 85, 979-987 (2016).-   G. Alvarez-Bolado, F. A. Paul, S. Blaess, Sonic hedgehog lineage in    the mouse hypothalamus: from progenitor domains to hypothalamic    regions. Neural development 7, 4 (2012).-   S. Blaess, N. Szabo, R. Haddad-Tovolli, X. Zhou, G. Alvarez-Bolado,    Sonic hedgehog signaling in the development of the mouse    hypothalamus. Front Neuroanat 8, 156 (2014).-   E. O. Mazzoni et al., Synergistic binding of transcription factors    to cell-specific enhancers programs motor neuron identity. Nat    Neurosci 16, 1219-1227 (2013).-   P. Arlotta, O. Hobert, Homeotic Transformations of Neuronal Cell    Identities. Trends Neurosci 38, 751-762 (2015).-   E. S. Deneris, O. Hobert, Maintenance of postmitotic neuronal cell    identity. Nat Neurosci 17, 899-907 (2014).-   T. J. Cleophas, A. H. Zwinderman, T. F. Cleophas, in Statistics    Applied to Clinical Trials. (Springer Netherlands, Dordrecht, 2006),    pp. 219-228.-   S. Wellek, M. Blettner, On the Proper Use of the Crossover Design in    Clinical Trials: Part 18 of a Series on Evaluation of Scientific    Publications. Deutsches Ärzteblatt International 109, 276-281    (2012).-   T. A. Louis, P. W. Lavori, J. C. I. Bailar, M. Polansky Crossover    and Self-Controlled Designs in Clinical Research. New England    Journal of Medicine 310, 24-31 (1984).-   E. M. Dykens, M. A. Maxwell, E. Pantino, R. Kossler, E. Roof,    Assessment of Hyperphagia in Prader-Willi Syndrome. Obesity 15,    1816-1826 (2007).-   S. R. Crawford et al., The International Development of The Modified    Hyperphagia Questionnaire. Value in Health 18, A761.-   J. M. MD, D. D. MD, A. Chen, T. E. Hughes, D. D. Kim, paper    presented at the Obesity Week 2014, Boston, Mass., 2014.-   R. J. Kuppens, S. H. Donze, A. C. S. Hokken-Koelega, Promising    effects of oxytocin on social and food-related behaviour in young    children with Prader-Willi syndrome: a randomized, double-blind,    controlled crossover trial. Clinical Endocrinology 85, 979-987    (2016).-   P. Gumus Balikcioglu et al., Macronutrient Regulation of Ghrelin and    Peptide Y Y in Pediatric Obesity and Prader-Willi Syndrome. The    Journal of Clinical Endocrinology & Metabolism 100, 3822-3831    (2015).-   R. Kaddurah-Daouk, R. Weinshilboum, N. on behalf of the    Pharmacometabolomics Research, Metabolomic Signatures for Drug    Response Phenotypes: Pharmacometabolomics Enables Precision    Medicine. Clinical Pharmacology & Therapeutics 98, 71-75 (2015).-   R. D. Beger et al., Metabolomics enables precision medicine: “A    White Paper, Community Perspective”. Metabolomics 12, 149 (2016).

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Numerous references,including patents and various publications, are cited and discussed inthe description of this invention. The citation and discussion of suchreferences is provided merely to clarify the description of the presentinvention and is not an admission that any reference is prior art to theinvention described herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety.Variations, modifications and other implementations of what is describedherein will occur to those of ordinary skill m the art without departingfrom the spirit and scope of the invention. While certain embodiments ofthe present invention have been shown and described, it will be obviousto those skilled in the art that changes and modifications may be madewithout departing from the spirit and scope of the invention. The matterset forth in the foregoing description and accompanying drawings isoffered by way of illustration only and not as a limitation. The actualscope of the invention is intended to be defined in the followingclaims.

1.-54. (canceled)
 55. A method for treating Prader-Willi Syndrome (PWS)comprising administering a phosphodiesterase 4 inhibitor (PDE4i) to asubject with PWS, in an amount that alleviates, eliminates or preventsone or more symptoms of PWS.
 56. The method of claim 55, wherein the oneor more symptoms comprises hyperphagia, reduced metabolic rate, obesity,hypogonadism, decreased growth hormone production, poor muscle tone,reduced stamina, reduced ability to focus, impaired cognition, anxiety,growth failure, reduced conversion of immature hormones to mature andactive forms, or diabetes.
 57. The method of claim 55, wherein the oneor more symptoms comprises hyperphagia.
 58. The method of claim 55,wherein the one or more symptoms comprises reduced conversion to matureand active forms of one or more of insulin, ghrelin, oxytocin, growthhormone releasing hormone (GHRH), brain-derived neurotrophic factor(BDNF) or gonadotropin.
 59. The method of claim 55, wherein the PDE4i isa selective PDE4 inhibitor.
 60. The method of claim 55, wherein themethod further comprises administering one or more additionaltherapeutic agents effective for treating or alleviating one or moresymptoms of PWS.
 61. The method of claim 55, wherein the method furthercomprises administering an oxytocin receptor agonist.
 62. The method ofclaim 55, wherein the method further comprises administering insulin, aninsulin receptor agonist, ghrelin, a ghrelin receptor agonist, GHRH, aGHRH receptor agonist, alpha-MSH, an alpha-MSH receptor agonist,oxytocin, an oxytocin receptor agonist, orexin, an orexin receptoragonist, BDNF, a BDNF receptor agonist, vasopressin, a vasopressinreceptor agonist, NPY, an NPY receptor agonist, AGRP, an AGRP receptoragonist, gonadotropin, a gonadotropin receptor against, or combinationsthereof.